The primary role of B cells, antibody production and antigen elimination, is controlled by the B cell receptor (BCR) complex. BCR crosslinking activates a recently discovered and powerful signaling system mediated by the ER membrane proteins, STIM1 and STIM2. Interacting directly with the PM, STIM proteins expose a reactive domain that avidly binds and traps a specialized family of channel proteins, Orai1, Orai2, and Orai3. The Orai1 channels are exceedingly selective Ca2+ channels that, upon direct binding to STIM sensors, become activated to conduct Ca2+ ions into the junctional cytosolic space. This highly controlled entry of Ca2+ is crucial for two reasons: (i) to replenish Ca2+ within the ER preventing cell stress from protein misfolding, and allowing Ca2+ release signals to be maintained; (ii) to provide longer term and spatially defined Ca2+ signals mediating control over transcription, growth, or apoptosis. The STIM-Orai signaling pathway has particular significance in B cells - the precise coordination of Ca2+ release and entry signals mediates oscillatory Ca2+ signals, the amplitude and duration of which determine how B cells respond to BCR antigen-binding to undergo either proliferation, anergy, or apoptosis. The work combines molecular, biophysical, and cellular approaches to study STIM and Orai proteins using the DT40 B cell line and HEK293 human kidney-derived cells. DT40 B cells retain functional BCR-coupled signaling machinery and we have lines in which each STIM and Orai protein is knocked out. Using these cells our three specific aims are: 1. To examine the distinct functional roles of STIM1 and STIM2 proteins in mediating Ca2+ entry signals. 2. To ascertain how STIM1 and STIM2 proteins interact with and control Orai Ca2+ channels. 3: To examine the STIM-Orai Ca2+ signaling microenvironment in B cells. The studies dissect a novel Ca2+ signaling process fundamentally connected to the BCR-coupled machinery, exerting crucial regulatory control over B cell function. The STIM-Orai signaling pathway and its central role in Ca2+ signal generation in B cells provides a novel and important pharmacological target. The size and duration of Ca2+ signals are primary determinants of B cell fate in response to BCR activ- ation - cell division, maintenance, or, in the case of self-recognition, cell death. Defining the mechanistic operation of this long-acting Ca2+ signaling pathway and examining its pharmacological modification by the borate, 2-APB, provides a target through which B cell function and development can be modified providing the potential to control major immunological diseases including primary B cell deficiencies, lymphoproliferative disorders such as chronic lymphocytic leukemia, and autoimmune diseases. PUBLIC HEALTH RELEVANCE: B cells, the crucial blood cells that produce antibodies and provide immunity, are fundamentally controlled by calcium signals generated by the binding of antigens to the B cell surface. An entirely new signaling pathway involving important regulatory proteins that move throughout the cell, trigger calcium signals that cause control of key B cell functions including growth and differentiation. These new signaling pathways provide vital new targets to pharmacologically control B cell function and development, providing the means to control major immunological diseases including lymphoproliferative disorders such as chronic lymphocytic leukemia and autoimmune diseases.